ABSTRACT Wild type (WT) RAS (H, N and K) GTPases hydrolyze GTP to GDP and cycle between an active, GTP bound, and an inactive, GDP-bound, state. This is mediated by guanine nucleotide exchange factors (GEFs, e.g. SOS), which catalyze the exchange of GDP for GTP, and GTPase activating proteins (GAPs, e.g. NF1 or RASA1), which potentiate a weak intrinsic GTPase activity. KRAS mutations comprise one of the most frequent activating alterations found in cancer patients. KRASG12C, in particular, is the most frequent KRAS mutation in lung cancer, a disease responsible for nearly 150,000 deaths each year in the US. Despite the prevalence of these mutations, no therapies that directly target this oncoprotein are currently available in the clinic. A recently identified binding pocket in KRASG12C has led to the discovery of compounds that potently inhibit the levels of KRAS-GTP and effector signaling by this oncoprotein. Such compounds now enable a novel approach to study the regulation and activity of the KRAS oncoprotein in cancer. In a recent Science article, we described the mechanism by which allele specific inhibitors suppress KRASG12C-signaling and cancer cell growth. These drugs trap the oncoprotein in its inactive state and prevent its reactivation by nucleotide exchange factors. Our work predicts that nucleotide exchange activity is inversely related to the kinetics and/or magnitude of inhibition. Based on this conceptual model and through a comprehensive effort integrating genetic, biochemical and proteomic approaches we will now study the regulation of KRASG12C and that of other KRAS oncoproteins in cancer cells and then identify optimal therapeutic modalities that can be carried forward in clinical trials. In aim 1 we will determine if these oncoproteins exist in an excitable state and if this affects their tumor-forming potential. In aim 2 we will use a novel assay to identify regulators of KRASG12C signaling and its inhibition by allele-specific drugs. In aim 3 we will investigate the effect of modulating KRAS activity on tumor growth and identify combination treatments with improved efficacy in patient-derived xenograft models. The impact of the proposed work centers on advancing our understanding of how KRAS oncoproteins are activated in cancer, providing insight into the mechanisms that govern sensitivity or resistance to the novel inhibitors and the identification of an optimal therapy to treat patients whose tumors harbor a KRASG12C mutation.